Isolation and Identification of Microorganisms from Fermented Using Black Bamboo (Gigantochloa Atroviolacea) and Sweet Bamboo (Gigantochloa Atter)

Gladys Delarosa Purnomo1), Lindayani2) and Laksmi Hartayanie2)

1) Students; Food Technology Department; Agricultural Technology Faculty; Soegijapranata Catholic University 2) Lecturer; Food Technology Department; Agricultural Technology Faculty; Soegijapranata Catholic University [email protected]

ABSTRACT

Fermented glutinous rice is a kind of traditional food from glutinous rice fermented with ragi. The typically containers used for fermentation glutinous rice are banana leaves, plastic bottles, etc. Some efforts to increase local knowledge or use natural resources from environment is very important, for the example is to use bamboo. Traditional Chinnese food was made using bamboo to get a spesific flavor, so in this research bamboo was used as a container of fermented glutinous rice. The purpose of this study was to identify the microorganisms found in fermented glutinous rice. There was a preliminary test (sensory evaluation) to determine the best ragi and packaging by 30 panelists. The results of sensory evaluation showed that local ragi tape NKL and black bamboo obtained from Ungaran were the best result. The methods of this study include chemical analysis (total sugars, total acids and pH) and microbiological analysis (isolation and identification of lactic acid , and khamir). Identification tests of lactic acid bacteria were done by characteristics morphology test (cell shape, gram staining and spore formation), catalase activity test, motility test, gas production test, growth capabilities test at various temperatures, pH and NaCl. Identification of lactic acid bacteria used API 20 STREP test kit. The results shown that isolate identified as Aerococcus viridans 3 and Aerococcus viridans 2. identification obtained 5 isolates from Aspergillus sp. and 2 isolate from Mucor sp. Yeast identification using API 20 C AUX identified as Saccharomyces cerevisiae 1 and Candida utilis.

Keyword: fermented glutinous rice, lactic acid bacteria, molds, yeast, ragi, bamboo

INTRODUCTION

The heterogeneous of ethnic and cultural diversity enriched with various types of traditional foods. The Indonesian traditional fermented foods such as , tempe, oncom, tape and saurkraut. One of fermented foods widely known is tape. Many types raw material that used to production tape in Indonesia, such as , sweet potato, sorghum, banana, and glutinous rice (black and white).

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Fermented Glutinous Rice (“Tape Ketan”) is a traditional food made from glutinous rice (Oryza sativa var glutinosa) and fermented with ragi. Cronk et al. (1977) explain that tape ketan is a fermented food, have sweet and sour taste and likely rice pasta contains little alcohol. According Lestario (1998) in Atmodjo (2006), good tape ketan have soft grain texture, sticky, sweet and slightly sour taste and contains little alcohol and not sting. In general, the process of making tape using anaerobic fermentation process. After rice blend with ragi, put into containers, then stored in sealed containers for 2-3 days at temperature 26-280C. Tape inculated with anaerobic fermentation have sweeter taste than aerobic fermentation, because microorganisms in tape ketan does not need many oxygen (Tarigan, 1988).

Tape ketan usually fermented in plastic containers. In specific areas, tape ketan wrapped with banana leaves. Some efforts to increase local knowledge or use natural resources in environment is very important, for example is bamboo. Bamboo is still limited to used, such as for structural materials and cooking for chinese food. Traditional Chinnese food used bamboo to get spesific flavor. So in this research wants to used bamboo as packaging in fermented tape ketan.

Fermentation process involves microorganisms to change components in foods from macro nutrient molecules into simple molecules. This foods become easy to digested and absorbed by intestine. Microorganisms involves in fermentation process are yeast, fungi and bacteria (Volk & wheeler, 1984). According Hesseltine et al. (1988), ragi tape have at least one species of yeast, one species mold (Mucor, Rhizopus, or Amylomyces) and one or two coccus bacteria in each sample. Gunadya & Between (1997) found, microorganisms in ragi tape such as Aspergillus, Saccharomyces, Candida, Hansenula and Acetobacter. According to Sujaya et al. (2001), Lactic Acid Bacteria that found in tape ketan are Weissella, Lactobacilli, Enterococci and Pediococci. Microorganisms in ragi tape can influenced microorganisms founded in tape ketan because ragi acts to start fermentation. There are many factors that influence fermentation process such as time, moisture, temperature, oxygen and substrates (Gaman and Sherrington, 1994) so microorganisms in fermented foods can be different from region to other region. The objectives in this research were to isolate and to identify microorganisms in tape ketan which fermented in black bamboo and sweet bamboo.

MATERIALS AND METHODS

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Making of Tape Ketan

Glutinous rice

Figure 1. Making Process of Tape Ketan

Preliminary Test

In preliminary test performed by sensory analysis with rating test hedonic to determine level of consumer preference tape ketan with variety local yeast. Testing by filling out questionnaires provided. The panelists must give score 1 (one) to 5 (five), which score one indicates the lowest value (strongly dislike) and score five indicates highest value (really like) (Resurrecion, 1998). Organoleptic parameters tested included flavor, aroma, texture and color. Panelists to be used is untrained panelists in UNIKA Soegijapranata students (30 people). There were 12 samples tested in preliminary test that is combination of packaging (black bamboo and sweet bamboo), ragi local brands (Na Kok Liong, Gedang and Sidojoyo), and concentrated ragi (0.2% and 0.3%). Preliminary tests to determine the best concentration ragi, ragi local brands and packaging to be used in main test.

Main Test

In main test, sample used is the best results sample from preliminary test, that is tape ketan saved in sweet bamboo, used NKL local ragi brand and the concentration is 0,2%.

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Microbiological analyzes conducted in 2 times and physicochemical analyzes conducted in 3 times. Physicochemical testing conducted in day 3, 5, 7 and 9. Tape Ketan

Figure 2. Main Research Flowchart Design

The methods of measuring total acid is titration method (Simbolon, 2008). Measurement pH using pH meter. And for total sugars using phenol method (Apriyantono et al., 1989). Isolation molds, yeast and LAB using same methods that is isolate colony until form a single colony. Colony purification method using spread plate method. Dilution for purification colony molds up to 10-5. Medium for mold is PDA with addition 0,1% chloramfenicol. Incubation for mold during 5 days at temperature 280C (Chiang et al., 2006; Lay, 1994). For purification method in LAB performed by diluting until 10-8. Media used is MRS with addition 1% of CaCO3. Incubation of BAL during 48 hours at temperature 370C (Chiang et al., 2006; Rahayu & Margino, 1997). In purification colony of yeast used MEA and dilution until 10-5. Incubation yeasts during 2-4 days at temperature 280C.

Identification of fungi was conducted by slides culture and incubated for 2-3 days. After incubation, molds observed using microscope and compairing morphological appearance with Introduction to Food Borne Fungi (Lay, 1994; Samson et al., 1984). Identification tests of LAB were done by morphology test (cell shape, gram staining and spore formation), catalase activity test, motility test, gas production test, and growth capabilities test at various temperatures (100C, 450C and 500C), pH (4.4 and 9.6) and NaCl (6.5% and 18%) (Lay, 1994; Rahayu & Margino, 1997; Sneath et al., 1984). Testing of species LAB do with API 20 Strep test kit (Pelinescu et al., 2009). Identification of yeast done by API 20 C AUX test kit (Kurtzman & Fell, 1998).

RESULTS AND DISCUSSION

Preliminary Test

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From the results of preliminary test known that local brand ragi NKL with concentration 0.2% and sweet bamboo are the best results (Table 1, Table 2). Then, NKL ragi local brands with concentration ragi 0.2% and sweet bamboo used for main test (physicochemical and isolation identification of microorganisms). Table 1. The Sensory of Tape Ketan based on NKL, Gedang and Sidojoyo

Concentration Merk ragi 0,20% 0,30% NKL 3,12 2,96 gedang 3,07 3,03 sidojoyo 3,03 3,01

Table 2. The Sensory of Tape Ketan based on Bamboo Species

Bamboo species Average hitam 3,03 legi 3,1111 Note: shown sample have the best results

Physicochemical Analyzes

Chemical analyses consist of total sugar, pH and total acid on days 3, 5, 7 and 9. Based on the results in Graph 1, known that total sugar contained in tape ketan decreased from day 3 to day 9. In results of pH, the value is going decreased from day 3 to day 9 (Graph 2). This is caused by lactic acid bacteria convert simple sugars such as glucose into organic acids, especially lactic acid (Holzapfel et al., 2003). And from total acid tested, known that total acid contained in tape ketan increased from day 3 to day 9 (Graph 3). At Graph pH and total acid tirtration obtained a relation, when total acid increased, pH value on tape ketan going decreased. This is in accordance with opinion from Ray & Bhunia (2007), higher acid content in product involve the pH value going lower.

Graph 1. Total Sugars Measured in Tape Ketan

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Graph 2. pH Values Measured in Tape Ketan

Graph 3. % Acid Tirtration Measured in Tape Ketan

Isolation and Identification Lactic Acid Bacteria

From the results of isolation and identification lactic acid bacteria, obtained 9 isolates form clear area in medium (Figure 3). Clear area due to formation of soluble Ca-lactate in medium (Djide & Wahyudin, 2008).

Figure 3. Isolate lactic acid bacteria formed single colony and form clear zone

Isolates purified until form a single colony and then identified further. First test in identification of lactic acid bacteria included morphological and biochemical tests (Table 3). Morphological tests included gram stain and spore formation. From identification, known that LAB are gram-positive bacteria (purple colored), a coccus shape and non forming spores (red colored). For biochemical tested included catalase activity, motility and gas production. From identification known that LAB has negative catalase activity

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because does not forming gas. Non-motile bacteria only growth along the line of inoculation, but if motile bacteria growth outside the line of inoculation. All isolates is homofermentatif bacteria because not produce bubble gas in durham (negative). Identification continued with physiological tests, this test performed to determine growth capabilities at temperatures (100C, 450C, and 500C), pH (4,4 and 9,6) and concentration of NaCl (6,5% and 18%) (Table 3). This physiological tests performed to determine genus of LAB. The results is varied for each isolate.

Table 3. Identification results of LAB

Temperature NaCl pH Gram Spore Gas 0 Isolate Catalase Motility ( C) (%) (%) staining formation Production 10 45 50 6,5 18 4,4 9,6

A Purple Red - - - + + - - - - + B Purple Red - - - + + + - - - - C Purple Red - - - - + + - - - + D Purple Red - - - + + + - - - + E Purple Red - - - + + + - - + + F Purple Red - + - - + + - - + + G Purple Red - - - + + - + - - + H Purple Red - - - - + + + - - + I Purple Red - - - + + + + - - + Notes : “+” = isolate growth (turbid) : “-“ = isolate not growth (clear)

Isolates have the best growth identified using API 20 Strep test kit. API 20 Strep test kit is a tool for identification LAB based on capability isolate to ferment various carbon sources (Pelinescu et al., 2009). The results are qualitatively observed by color changes according in tube respectively (Figure 4). After the isolates were identified, continued by analysis data using API WEB software. The results shown that isolates I identified as Aerococcus viridans 2 with level of significance 83,9 % and isolates G identified as Aerococcus viridans 3 with level of significance 96,9 %.

G

I

Figure 4. Identification Results of Isolate G and I using API 20 Strep test kit The 4 th International Conference of Indonesian Society Lactic Acid Bacteria (ISLAB). Yogyakarta, 25th – 26th January 2013 Page 7

Aerococcus viridans also known as Pediococcus urinae-equi have maximum temperature for growth at 420C with optimum temperature growth at 25-300C, start growth at medium agar with pH 6,5 – 7,0 (Wood & Holzapfel, 1995) and have good growth in pH 9,6 (Rahayu & Margino, 1997; Axelsson, 2004). A. viridans have coccus cell shape in pair and tetrad, gram-positive, catalase negative, microaerophilic and homofermentatif. These bacteria can ferment sugar and produce acid 0.5 - 0.9%, especially lactic acid and can growth in saline solution 5.5% (Frazier & Westhoff, 1988).

Isolation and Identification Molds

From isolation results obtained 8 single colony isolate were further identified. The identification process is carried out morphological observation at 10x and 40x magnifications microscope. Then, observations results compared with literature Introduction to Food Borne Fungi (Samson et al., 1984). From 7 isolates were identified, known that five isolates identified as Aspergillus sp. and two isolates identified as Mucor sp. Identification of molds only can be conducted until genus, by morphological observation molds with microscope (Table 4).

Table 4. Identification Results of Molds in Tape Ketan

Isolate Sporangium Sporangiospore Conidiospores Phialides Vesicle Columnella Genus 1 + - - - - - Mucor sp. 1 2 - - + + + - Aspergillus sp. 1 3 - - + + + - Aspergillus sp. 2 4 - - + + + - Aspergillus sp. 3 5 + + - - - + Mucor sp. 2 6 - - + - + - Aspergillus sp. 4 7 - - + + + - Aspergillus sp. 5

Aspergillus is a molds that have positive and negative impact on economy, industry, agriculture and health (Bennett, 2009). Aspergillus sp. included in division Amastigomycota, class Ascomycetes, ordo Eurotiales, family Eurotiaceae and genus Aspergillus (Bendre & Kumar, 2010). Most of class from Ascomycetes, proliferate with conidia (asexual spores), non motile, and formed by sporogeneus cells at the end of hyphae (Alexopaulus & Mims, 1979).

According Gandjar et al., (2006), Mucor including as mold that can produce enzyme amylase. Enzyme amylase is an extracellular enzyme, that is produced in cell and released into fermentation medium surrounding the cells, so can hydrolyze macromolecules. Macromolecules such as will be degraded to glucose by amiloglukosidase (Dung et al., 2006 in Tamang & Kailasapathy, 2010). This is caused the total glucose in fermentation to high, reach 43%. Mucor can grow rapidly, both hyphae and proliferation in number of sporangiophore (Bagyaraj & Arpana, 2006). Mucor growth in pH 3,9 - 6,2 and

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in some species can growth at pH of 6,8 - 7,9 (Alves et al., 2005). This is in accordance with research, Mucor can growth in tape ketan with pH 4,51 (Graph 2).

Isolation and Identification Yeasts

Isolation of yeast from tape ketan obtained 10 single colony isolates (Figure 5). The single colony isolates identified further using API 20 C AUX. API 20 C AUX used to determine species of yeasts based on physiological characteristics. API 20 C AUX Strip consists of 20 tubes containing dehydrated substrates that can be used to see grow capabilities isolate at 19 test assimilation. Tubes were inoculated with semi-solid medium and yeast only can grow if they can utilize any substrate as single carbon source. The results observed qualitatively by color changes (Figure 6). Positive result indicated colors change becomes more turbid and negative results indicated by no color change (clear) (Kurtzman & Fell, 1998).

Figure 5. Isolate Yeast Isolated from Fermented Tape Ketan using Sweet Bamboo

Figure 6. Identification Results of Isolate 2A4 and 4B4 using API 20 C AUX

Isolates have the best growth were tested using API 20 C AUX, that is isolates 2A4 and 4B4. After isolates were identified using API 20 C AUX, continued with analysis data using API WEB software. The identification results shown that isolates 2A4 identified as Saccharomyces cereviseae 1 with level of significance 96,2 % and isolate 4B4 identified as Candida utilis with level of significance 96,4 %.

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Yeast play important roles in because produces enzymes that help chemical reactions, such as formation of alcohol and sugar (Battcock & Azam-Ali, 1998). Yeasts produce ethyl alcohol and carbon dioxide from simple sugars like glucose and fructose (Farnworth, 2005). This is in accordance with the research, total sugars contained in tape ketan decreased on day 3 to day 9. This is because all simple sugars in tape ketan converted into alcohol and carbon dioxide. Candida utilis is an unicellular eukaryotic organisms, that are asporogenous yeasts produce pseudomiselium (Jay, 1986). C. utilis is one of main microorganisms in ragi used for fermentation ethanol (Bailey, 1986).

Saccharomyces cerevisiae is a yeast that is widely used in industrial fermentation (Rahayu et al., 2005). S. cereviseae have diploid cell form, which is oval shaped with size larger than haploid cells have round shape. Yeast have various size with length 1-5 μm to 20-50 μm and wide 1-10 μm (Jutono, 1975). S. cereviseae have pH optimum growth from 4.0 to 5.0 and minimum pH 2.0 to 2.4 (Banwart, 1979). This is appropriate with research, S. cereviseae can growth at pH 4,51 (Figure 4).

CONCLUSIONS

From isolation and identification in tape ketan during 3 days of fermentation, obtained two species of lactic acid bacteria were identified, namely Aerococcus viridans 2 and Aerococcus viridans 3, five species from Aspergillus sp., two isolates from Mucor sp, and two kinds of yeast, namely Saccharomyces cereviseae 1 and Candida utilis.

REFERENCES

Alves, M. H., Galba, M. De Campos-Takaki, Kaoru, O., Ine, H. F. P. & Adauto I. M. 2005. Detection of Extracellular Protease in Mucor Species. Rev Iberoam Micol Vol 22: 114-117.

Atmodjo, K. 2006. Pengaruh Variasi Beras Ketan (Oryza sativa var. Glutinosa L.) dan Suhu fermentasi Terhadap produksi Alkohol. Biota XI (3): 152-158.

Chiang Y. W., Chye, F. Y., Mohd Ismail, A. 2006. Microbial Diversity and Proximate Composition of , A Sabah’s Fermented Beverage. Malaysian Journal of Microbiology Vol. 2(1): 1-6.

Cronk, T. C.; K. H. Steinkraus; L. R. Hackler & L. R. Mattick. 1977. Indonesian Tape Ketan Fermentation. Applied and Environmental Microbiology Vol. 33 (5): 1067- 1073.

Frazier, W. C. and Westhof, D. C. 1988. Food Microbiology. Singapore: McGraw Hill Book Company.

Kurtzman, C. P. and J. W. Fell. 1998. A Taxonomic Study. 4th Edition. Elsevier Science, Amsterdam.

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Rahayu, E. S. dan S. Margino. 1997. Bakteri Asam Laktat: Isolasi dan Identifikasi. PAU Pangan dan Gizi. Universitas Gajah Mada. Yogyakarta.

Ray, B. and A. Bhunia. 2007. Fundamental Food Microbiology. 4th Edition. CRC Press. United States of America.

Samson, R. A.; E. S. Hoekstra; & C. A. N. Van Oorschot. 1995. Introduction to Foodborne Fungi. Central Guerau Voor Schinmelculture. Baarn.

Sneath, P. H. A., N. S. Mair, M. E., Sharpe, J. G. Holt. 1984. Bergeys Manual of Systematic Bacteriology. Vol 2. Williams & Wilkins. Baltimore.

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